Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease

Endothelial protein C receptor (EPCR) and thrombomodulin (TM) are expressed at high levels in the resting microvasculature and convert protein C (PC) into its activated form, which is a potent anticoagulant and antiinflammatory molecule. Here we provide evidence that in Crohn disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), there was loss of expression of endothelial EPCR and TM, which in turns caused impairment of PC activation by the inflamed mucosal microvasculature. In isolated human intestinal endothelial cells, administration of recombinant activated PC had a potent antiinflammatory effect, as demonstrated by downregulated cytokine-dependent cell adhesion molecule expression and chemokine production as well as inhibited leukocyte adhesion. In vivo, administration of activated PC was therapeutically effective in ameliorating experimental colitis as evidenced by reduced weight loss, disease activity index, and histological colitis scores as well as inhibited leukocyte adhesion to the inflamed intestinal vessels. The results suggest that the PC pathway represents a new system crucially involved in governing intestinal homeostasis mediated by the mucosal microvasculature. Restoring the PC pathway may represent a new therapeutic approach to suppress intestinal inflammation in IBD.     

Mice with a severe deficiency in protein C display prothrombotic and proinflammatory phenotypes and compromised maternal reproductive capabilities

Anticoagulant protein C (PC) is important not only for maintenance of normal hemostasis, but also for regulating the host immune response during inflammation. Because mice with a designed total genetic deficiency in PC (PC-/- mice) die soon after birth, attempts to dissect PC function in various coagulation/inflammation-based pathologies through use of mice with less than 50% of normal PC levels have not been successful to date. In the current investigation, we have used a novel transgenic strategy to generate different mouse models expressing 1-18% of normal PC levels. In contrast to PC-/- mice, mice with only partial PC deficiency survived beyond birth and also developed thrombosis and inflammation. The onset and severity of these phenotypes vary significantly and are strongly dependent on plasma PC levels. Our findings additionally provide the first evidence that maternal PC is vital for sustaining pregnancy beyond 7.5 days postcoitum, likely by regulating the balance of coagulation and inflammation during trophoblast invasion. These low PC-expressing transgenic mouse lines provide novel animal models that can be used to elucidate the importance of PC in maintenance of the organism and in disease.     

Treatment of sepsis-induced acquired protein C deficiency reverses Angiotensin-converting enzyme-2 inhibition and decreases pulmonary inflammatory response

The protein C (PC) pathway plays an important role in vascular and immune function, and acquired deficiency during sepsis is associated with increased mortality in both animal models and in clinical studies. However, the association of acquired PC deficiency with the pathophysiology of lung injury is unclear. We hypothesized that low PC induced by sepsis would associate with increased pulmonary injury and that replacement with activated protein C (APC) would reverse the activation of pathways associated with injury. Using a cecal ligation and puncture (CLP) model of polymicrobial sepsis, we examined the role of acquired PC deficiency on acute lung injury assessed by analyzing changes in pulmonary pathology, chemokine response, inducible nitric-oxide synthase (iNOS), and the angiotensin pathway. Acquired PC deficiency was strongly associated with an increase in lung inflammation and drivers of pulmonary injury, including angiotensin (Ang) II, thymus and activation-regulated chemokine, plasminogen activator inhibitor (PAI)-1, and iNOS. In contrast, the protective factor angiotensin-converting enzyme (ACE)-2 was significantly suppressed in animals with acquired PC deficiency. The endothelial protein C receptor, required for the cytoprotective signaling of APC, was significantly increased post-CLP, suggesting a compensatory up-regulation of the signaling receptor. Treatment of septic animals with APC reduced pulmonary pathology, suppressed the macrophage inflammatory protein family chemokine response, iNOS expression, and PAI-1 activity and up-regulated ACE-2 expression with concomitant reduction in AngII peptide. These data demonstrate a clear link between acquired PC deficiency and pulmonary inflammatory response in the rat sepsis model and provide support for the concept of APC as a replacement therapy in acute lung injury associated with acquired PC deficiency.     

Science review: Role of coagulation protease cascades in sepsis

Cellular signaling by proteases of the blood coagulation cascade through members of the protease-activated receptor (PAR) family can profoundly impact on the inflammatory balance in sepsis. The coagulation initiation reaction on tissue factor expressing cells signals through PAR1 and PAR2, leading to enhanced inflammation. The anticoagulant protein C pathway has potent anti-inflammatory effects, and activated protein C signals through PAR1 upon binding to the endothelial protein C receptor. Activation of the coagulation cascade and the downstream endothelial cell localized anticoagulant pathway thus have opposing effects on systemic inflammation. This dichotomy is of relevance for the interpretation of preclinical and clinical data that document nonuniform responses to anticoagulant strategies in sepsis therapy.     

Endothelium and regulation of coagulation

Endothelial cells form the luminal vascular surface and thus have a central role in the regulation of coagulation. One important way in which endothelial cells control the clotting system is by regulating the expression of binding sites for anticoagulant and procoagulant factors on the cell surface. In the quiescent state, endothelial cells maintain blood fluidity by promoting the activity of numerous anticoagulant pathways, including the protein C/protein S pathway. After activation, as can be brought about by cytokines, the balance of endothelial properties can be tipped to favor clot formation through coordinated induction of procoagulant and suppression of anticoagulant mechanisms. Tumor necrosis factor suppresses the endothelial anticoagulant cofactor thrombomodulin and induces expression of the procoagulant cofactor tissue factor. Working in concert, these changes can allow fibrin formation to proceed in an inflamed focus but maintain blood fluidity in the surrounding area of normal vasculature. Recent studies suggest that similar changes in endothelial coagulant properties can be induced by advanced glycosylation end products, proteins modified by glucose that accumulate in the vasculature at a rapid rate in diabetic subjects, indicating the potential relevance of these mechanisms in diabetic vascular disease.     

Activation Mechanism of Anticoagulant Protein C in Large Blood Vessels Involving the Endothelial Cell Protein C Receptor

Protein C is an important regulatory mechanism of blood coagulation. Protein C functions as an anticoagulant when converted to the active serine protease form on the endothelial cell surface. Thrombomodulin (TM), an endothelial cell surface receptor specific for thrombin, has been identified as an essential component for protein C activation. Although protein C can be activated directly by the thrombin–TM complex, the conversion is known as a relatively low-affinity reaction. Therefore, protein C activation has been believed to occur only in microcirculation. On the other hand, we have identified and cloned a novel endothelial cell surface receptor (EPCR) that is capable of high-affinity binding of protein C and activated protein C. In this study, we demonstrate the constitutive, endothelial cell–specific expression of EPCR in vivo. Abundant expression was particularly detected in the aorta and large arteries. In vitro cultured, arterial endothelial cells were also found to express abundant EPCR and were capable of promoting significant levels of protein C activation. EPCR was found to greatly accelerate protein C activation by examining functional activity in transfected cell lines expressing EPCR and/or TM. EPCR decreased the dissociation constant and increased the maximum velocity for protein C activation mediated by the thrombin–TM complex. By these mechanisms, EPCR appears to enable significant levels of protein C activation in large vessels. These results suggest that the protein C anticoagulation pathway is important for the regulation of blood coagulation not only in microvessels but also in large vessels.     

Protein C pathway in sepsis

The goals of this chapter are to provide a brief review of the biology of the protein C pathway and some of the features of the pathway that make it uniquely positioned to control microvascular coagulation and control the acute inflammatory response. Activated protein C works as an antithrombotic agent by inactivating factors Va and VIIIa. It is particularly effective at preventing microvascular thrombosis. Platelets may provide a margin of safety for activated protein C as an antithrombotic. Approximately 25% of the factor V/Va in plasma is contained within the platelet and hence resistant to time dependent inactivation by activated protein C. In addition, factor Va bound to the platelet surface is relatively resistant to inactivation by activated protein C. Activated protein C also facilitates clot lysis by inhibiting plasminogen activator inhibitor 1, a process that is accelerated markedly by vitronectin. Inflammatory cytokines like tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) downregulate two key components of the protein C activation complex, thrombomodulin and the endothelial cell protein C receptor resulting in decreased protein C activation. Activated protein C in turn has been shown in several animal models and in vitro to inhibit TNF elaboration in response to endotoxin. This inhibition appears to be due to diminished nuclear factor kappaB (NF kappaB) expression and nuclear translocation. Activated protein C has been shown to reduce the rate of death due to severe sepsis. This reduction may be due to both the anticoagulant effects as demonstrated by a reduction in D-dimer and inflammatory effects as demonstrated by a reduction in interleukin 6.     

凝血酶调节蛋白基因对脐静脉内皮细胞抗凝功能的调控

目的将含入凝血酶调节蛋白(hTM)基因的真核表达载体质粒 pcDNA3.1/hTM 转染体外培养的脐静脉内皮细胞(HUVECs),观察外源凝血酶调节蛋白的表达及其所致的 HUVECs 抗凝功能的改变。方法由阳离子脂质体介导将 pcDNA3.1/hTM 质粒转入内皮细胞中,半定量 RT-PCR 测定各组 hTM mRNA 的表达强度;免疫组化法检测 hTM 在内皮细胞膜上的表达;测定各组内皮细胞对蛋白 C(PC)的激活;半自动凝血仪测定内皮细胞-PC 反应液对正常血浆活化部分凝血活酶时间(APTT)和凝血酶原时间(PT)的影响。结果 HUVECs pcDNA3.1/hTM 质粒转染率约为10%,TM mRNA 和 TM 蛋白的表达强度都有明显提高。重组质粒转染组、空载质粒组及未转染组 PC 反应液中活化蛋白 C(acti-vated protein C,APC)的含量分别是(2.80±0.43)μg/ml、(0.75±0.08)μg/ml、(0.85±0.11)μg/ml。APTT 值在重组质粒转染组、空载质粒组、未转染组、未激活 PC 组和正常对照组中分别为(51.68±2.73)s、(38.38±2.44)s、(39.65±2.39)s、(33.93±1.73)s 和(34.60±1.86)s。PT 值在各组中分别为(21.89±1.66)s、(20.56±1.74)s、(20.42±2.04)s、(19.57±1.36)s 和(20.16±1.35)s。结论pcDNA3.1/hTM 质粒能被导入内皮细胞中,表达的 TM 分子具有生物学活性,能明显提高对 PC 的激活。激活的 PC 能明显抑制血浆内源性凝血途径使 APTT 延长,也使外源性凝血途径受到轻度抑制。     

Soluble thrombomodulin activity and soluble thrombomodulin antigen in plasma.

Endothelial cell membrane-bound thrombomodulin (TM) plays a critical role as a cofactor in the protein C pathway, important in regulating coagulation as well as inflammation. Heterogeneous soluble TM fragments circulate in the plasma and are found at increased levels in various diseases such as cardiovascular disease and diabetes, and in ischemic and/or inflammatory endothelial injuries. The anticoagulant function of these soluble fragments has not been measured in healthy individuals or in patients. Using an immobilized monoclonal antibody against TM and a microtiter plate format, an assay was designed to capture the soluble TM fragments in plasma and measure their cofactor activity in the thrombin-mediated activation of protein C. In addition, soluble TM antigen levels were measured by enzyme-linked immunosorbent assay. Both assays were used to investigate a group of healthy blood donors. TM fragments released into plasma were shown to retain significant cofactor activity, and reference intervals for healthy men and women were established. Furthermore, a statistically significant correlation was observed between soluble TM antigen levels and soluble TM cofactor activity. This notwithstanding, soluble TM activity only accounted for a minor part of all variation in soluble TM antigen levels (R2 = 22% in men and R2 = 16% in women).     

Modulation of endothelial cell hemostatic properties by tumor necrosis factor

Tumor necrosis factor/cachectin (TNF) is a mediator of the septic state, which involves diffuse abnormalities of coagulation throughout the vasculature. Since previous studies have shown that endothelial cells can play an active role in coagulation, we wished to determine whether TNF could modulate endothelial cell hemostatic properties. Incubation of purified recombinant TNF with cultured endothelial cells resulted in a time- and dose-dependent acquisition of tissue factor procoagulant activity. Concomitant with enhanced procoagulant activity, TNF also suppressed endothelial cell cofactor activity for the anticoagulant protein C pathway; both thrombin-mediated protein C activation and formation of functional activated protein C-protein S complex on the cell surface were considerably attenuated. Comparable concentrations of TNF (half-maximal affect at approximately 50 pM) and incubation times (half-maximal affect by 4 h after addition to cultures) were required for each of these changes in endothelial cell coagulant properties. This unidirectional shift in cell surface hemostatic properties favoring promotion of clot formation indicates that, in addition to leukocyte procoagulants, endothelium can potentially be instrumental in the pathogenesis of the thrombotic state associated with inflammatory and malignant disorders.     

Specificity of coagulation factor signaling

Summary.  Coagulation serine proteases signal through protease-activated receptors (PARs). Thrombin-dependent PAR signaling on platelets is essential for the hemostatic response and vascular thrombosis, but regulation of inflammation by PAR signaling is now recognized as an important aspect of the pro- and anti-coagulant pathways. In tissue factor (TF)-dependent initiation of coagulation, factor (F) Xa is the PAR-1 or PAR-2-activating protease when associated with the transient TF–FVIIa–FXa complex. In the anticoagulant protein C (PC) pathway, the thrombin–thrombomodulin complex activates PC bound to the endothelial cell PC receptor (EPCR), which functions as a required coreceptor for activated PC-mediated signaling through endothelial cell PAR-1. Thus, the pro- and anti-inflammatory receptor cascades are mechanistically coupled to immediate cell signaling, which precedes systemic coagulant or anticoagulant effects. In contrast to the substrate-like recognition of PARs by thrombin, TF- or EPCR-targeted activation of PARs generates cell-type specificity, PAR selectivity and protease receptor cosignaling with the G-protein-coupled PAR response. Protease receptors are thus major determinants of the biological outcome of coagulation factor signaling on vascular cells.     

Molecular recognition in the protein C anticoagulant pathway

Summary.  The protein C (PC) anticoagulant system provides specific and efficient control of blood coagulation. The system comprises circulating or membrane-bound protein components that take part in complicated multimolecular protein complexes being assembled on specific cellular phospholipid membranes. Each of the participating proteins is composed of multiple domains, many of which are known at the level of their three-dimensional structures. The key component of the PC system, the vitamin K-dependent PC, circulates in blood as zymogen to an anticoagulant serine protease. Activation is achieved on the surface of endothelial cells by thrombin bound to the membrane protein thrombomodulin. The endothelial PC receptor binds the Gla domain of PC and stimulates the activation. Activated PC (APC) modulates the activity of blood coagulation by specific proteolytic cleavages of a limited number of peptide bonds in factor (F)VIIIa and FVa, cofactors in the activation of FX and prothrombin, respectively. These reactions occur on the surface of negatively charged phospholipid membranes and are stimulated by the vitamin K-dependent protein S. Regulation of FVIIIa activity by APC is stimulated not only by protein S but also by FV, which, like thrombin, is a Janus-faced protein with both pro- and anticoagulant potential. However, whereas the properties of thrombin are modulated by protein–protein interactions, the specificity of FV function is governed by proteolysis by pro- or anti-coagulant enzymes. The molecular recognition of the PC system is beginning to be unravelled and provides insights into a fascinating and intricate molecular scenario.     

Multiple receptor-mediated functions of activated protein C

The central effector protease of the protein C pathway, activated protein C (APC), interacts with the endothelial cell protein C receptor, with protease activated receptors (PAR), the apolipoprotein E2 receptor, and integrins to exert multiple effects on haemostasis and immune cell function. Such receptor interactions modify the activation of PC and determine the biological response to endogenous and therapeutically administered APC. This review summarizes the current knowledge about interactions of APC with cell surface-associated receptors, novel substrates such as histones and tissue factor pathway inhibitor, and their implications for the biologic function of APC in the control of coagulation and inflammation.     

Protein C pathway in sepsis

The goals of this chapter are to provide a brief review of the biology of the protein C pathway and some of the features of the pathway that make it uniquely positioned to control microvascular coagulation and control the acute inflammatory response. Activated protein C works as an antithrombotic agent by inactivating factors Va and VIIIa. It is particularly effective at preventing microvascular thrombosis. Platelets may provide a margin of safety for activated protein C as an antithrombotic. Approximately 25% of the factor V/Va in plasma is contained within the platelet and hence resistant to time dependent inactivation by activated protein C. In addition, factor Va bound to the platelet surface is relatively resistant to inactivation by activated protein C. Activated protein C also facilitates clot lysis by inhibiting plasminogen activator inhibitor 1, a process that is accelerated markedly by vitronectin. Inflammatory cytokines like tumor necrosis factor &#102 (TNF &#102 ) and interleukin1 &#103 (IL-1 &#103 ) downregulate two key components of the protein C activation complex, thrombomodulin and the endothelial cell protein C receptor resulting in decreased protein C activation. Activated protein C in turn has been shown in several animal models and in vitro to inhibit TNF elaboration in response to endotoxin. This inhibition appears to be due to diminished nuclear factor &#115 B (NF &#115 B) expression and nuclear translocation. Activated protein C has been shown to reduce the rate of death due to severe sepsis. This reduction may be due to both the anticoagulant effects as demonstrated by a reduction in D-dimer and inflammatory effects as demonstrated by a reduction in interleukin 6.     

脑内水通道蛋白4表达调节的研究进展

脑内水通道蛋白4（AQP4）在细胞分化不同阶段表达情况不同，内皮细胞也影响其表达分布；星形胶质细胞及其微环境的渗透压、氨浓度、氧分压、温度以及一些其他因素如激素及肽类、外源性物质铅、补体抑制剂、脂多糖等的存在都会影响AQP4表达。但脑内AQP4表达的调节机制不十分清楚，与之相关的有蛋白的相互作用、蛋白激酶C（PKC）磷酸化途径、丝裂原激活的蛋白激酶信号转导途径、钙信号途径、转录因子活化等，其中研究最多的是PKC通过磷酸化抑制AQP4的活性。     

The normal role of Activated Protein C in maintaining homeostasis and its relevance to critical illness

Thrombin is a multifunctional protein, with procoagulant, inflammatory and anticoagulant effects. Binding of thrombin to thrombomodulin results in activation of Protein C and initiation of the Activated Protein C anticoagulant pathway, a process that is augmented by the endothelial cell Protein C receptor (EPCR). Activated Protein C has demonstrated antithrombotic, anti-inflammatory, and profibrinolytic properties. Its antithrombotic activity is particularly important in the microcirculation, and Protein C deficiency is associated with microvascular thrombosis. Activated Protein C has also been shown to modulate inflammation. When the level of thrombomodulin or Protein C is reduced in sepsis there is a vicious cycle of coagulation and inflammation, with potentially lethal consequences. In vitro studies and animal models have shown that Activated Protein C blunts the inflammatory and coagulant response to sepsis through a variety of mechanisms.     

Thrombomodulin: a linker of coagulation and fibrinolysis and predictor of risk of arterial thrombosis

Thrombomodulin (TM) binds thrombin, changes thrombin conformation and allows thrombin to activate protein C and thrombin-activatable fibrinolysis inhibitor (TAFI). Activated protein C and TAFI inhibit coagulation and fibrinolysis, respectively. TM is, thus, a linker of endogenous control of coagulation and fibrinolysis. Plasma soluble TM, cleaved products of cellular TM, also have anticoagulant and antifibrinolytic properties. TM plays an important role in thromboresistance. Prospective studies show that a high plasma TM level is associated with a low risk of developing coronary heart disease. The plasma TM level may reflect the level of endothelial TM expression. TM expression levels are influenced by multiple gene polymorphisms. Several of the polymorphisms are probably associated with coronary heart disease.     

Involvement of Akt and endothelial nitric oxide synthase in ventilation-induced neutrophil infiltration: a prospective,controlled animal experiment

Introduction  

Positive pressure ventilation with large tidal volumes has been shown to cause release of cytokines, including macrophage inflammatory protein-2 (MIP-2), a functional equivalent of human IL-8, and neutrophil infiltration. Hyperoxia has been shown to increase ventilator-induced lung injury, but the mechanisms regulating interaction between a large tidal volume and hyperoxia are unclear. We hypothesized that large tidal volume ventilation using hyperoxia would increase MIP-2 production and neutrophil infiltration via the serine/threonine kinase/protein kinase B (Akt) pathway and the endothelial nitric oxide synthase (eNOS) pathway.     

Loxosceles spider venom induces the release of thrombomodulin and endothelial protein C receptor: implications for the pathogenesis of intravascular coagulation as observed in loxoscelism

BACKGROUND: The venom of the spider Loxosceles can cause both local and systemic effects including disseminated intravascular coagulation. AIM: The aim of this study was to investigate the effects of the venom of Loxosceles intermedia (L. intermedia) and the purified Sphingomyelinase D (SMaseD) toxin upon the Protein C (PC) natural anticoagulant pathway. RESULTS: Both the venom and e purified SMaseD reduced the cell surface expression of thrombomodulin (TM) and Endothelial PC Receptor on endothelial cells in culture. The reduction of cell surface expression was caused by cleavage from the cell surface mediated by activation of an endogenous metalloproteinase. Reduction of TM and Endothelial PC Receptor on the surface of these cells resulted in an impaired ability of the cells to assist in the thrombin-induced activation of PC. CONCLUSION: This novel observation gives further insight into the mechanisms of the pathology induced by venom from Loxosceles spiders and may aid the development of a suitable therapy.